In recent years, as a result of environmental problems such as global warming and atmospheric pollution, as well as concerns related to energy supplies for transportation, including the dramatic increase in the cost of crude oil and the expectation of depletion of crude oil sources in the near future (peak oil), the development of alternative energy sources to oil has become an extremely important issue. Plant biomass or lignocellulose is the most plentiful renewable energy source on earth, and holds great promise as an alternative energy source to oil. The main component of plant biomass dry weight is lignocellulose, which is composed of polysaccharides such as cellulose and hemicellulose, and lignin. For example, polysaccharides can be hydrolyzed by a glycoside hydrolase such as a cellulase or hemicellulase to form monosaccharides such as glucose and xylose, which can then be used as biofuels or the raw materials for chemical products.
Lignocellulose is recalcitrant due to its highly complex structure, and is difficult to degrade or hydrolyze with a single glycoside hydrolase. The complete hydrolysis of lignocellulose generally requires three types of enzymes, namely an endoglucanase (cellulase or endo-1,4-β-D-glucanase, EC 3.2.1.4), an exo-type cellobiohydrolase (1,4-β-cellobiosidase or cellobiohydrolase, EC 3.2.1.91, EC 3.2.1.176), and a β-glucosidase (EC 3.2.1.21), and it is thought that the addition of a further plurality of enzymes including the hemicellulase xylanase (endo-1,4-β-xylanase, EC 3.2.1.8) and other plant cell wall-degrading enzymes such as β-xylosidase (EC 3.2.1.37) is also necessary.
In conventional bioethanol production using lignocellulose as a starting resource, hydrolysis processes using high solid loading (30 to 60% solid loading) have been tested with the aim of achieving a more energy-efficient conversion to ethanol. However, in this type of lignocellulose enzymatic hydrolysis using high solid loading, the viscosity of the hydrolyzed biomass solution is high, and the hydrolysis reaction of the lignocellulose tends to proceed poorly. Accordingly, by using a thermostable enzyme and performing the enzymatic hydrolysis process at a high temperature, for example 75° C. or higher, the rate of the hydrolysis reaction can be increased, and the viscosity of the hydrolyzed biomass solution can be reduced, which is expected to enable a shortening of the hydrolysis reaction time and a reduction in the amount of enzyme required. As a result, for all of the various glycoside hydrolases, the development of enzymes having superior thermal stability is very desirable.
When cellulose is hydrolyzed by a cellobiohydrolase, the disaccharide cellobiose is the main product. Cellobiohydrolases include some types which initiate hydrolysis from the reducing ends of cellulose (such as cellobiohydrolases belonging to the GH7 and GH48 families and the like), and some types which initiate hydrolysis from the non-reducing ends (such as cellobiohydrolases belonging to the GHS, GH6 and GH9 families and the like), and it is known that if the two types are used in combination, then the cellulose degradation activity is superior to that observed when either type is used alone (for example, see Non-Patent Document 1). Among cellobiohydrolases which initiate hydrolysis from the non-reducing ends of cellulose, a cellobiohydrolase of the GH6 family having an optimum temperature exceeding 75° C. has been reported (for example, see Patent Document 1).
However, in the case of cellobiohydrolases which initiate hydrolysis from the reducing ends, few enzymes of high thermal stability are known, although in the case of cellobiohydrolases belonging to the GH7 family, cellobiohydrolases have been isolated from the thermophilic filamentous fungi Chaetomium thermophilum (for example, see Non-Patent Document 2) and Thermoascus aurantiacus (for example, see Non-Patent Document 3) with optimum temperatures of 75° C. and 65° C. respectively. Further, in terms of cellobiohydrolases belonging to the GH48 family, Ce148A has been isolated from the thermophilic actinomycete Thermobifida furca (for example, see Non-Patent Document 4), and has an optimum temperature of about 60° C.